Genetic regulation of testosterone level in overweight males from the Kazakh population and its association with hypogonadism

Male hypogonadism and erectile dysfunction in different populations are associated with excess body weight. A key aspect in most studies is the metabolism of sexual hormones, primarily testosterone. At the same time, the binding protein sex hormone binding globulin (SHBG) can play a large role, as it determines the ratio of total and bioavailable testosterone in blood, i.e. both the hormone content and level of its production. Recent research has identified common mutations that affect SHBG levels, such as the rs727428 polymorphic locus, which is associated with alterations in histone protein function, affecting the regulation of ribonucleic acid (RNA) protein SHBG synthesis. Similar relationships have been observed for prevalent mutations, including rs5934505 and rs10822184, in diverse populations. This study involved 300 individuals of Kazakh nationality from the Eastern Kazakhstan region, examining three polymorphic variants of the SHBG gene (rs727428, rs5934505, and rs10822184). The participants were categorized into three groups: individuals with hypogonadism and obesity (group 1, n=85), those with excess body weight but no hypogonadism (group 2, n=70), and individuals with neither excess body weight nor hypogonadism (group 3, n=145). The frequency of mutant gene alleles impacting GPS (SHBG) synthesis in the Kazakh population was notably high, comparable to European and South-East Asian populations. However, the association between excess body weight and these mutations exhibited varying patterns. Hypogonadism was linked to decreased GPS levels, strongly correlating with total testosterone but not bioavailable testosterone. The retention of sexual functions in overweight men was not always directly related to BMI levels and GPS concentrations.


INTRODUCTION
Testosterone concentration decreases with age as part of a natural physiological process [1].Androgens are one of the key factors affecting the distribution of fat in a male body and are featured by the deposition of adipose tissue, mainly in the abdominal region.This can be a risk factor for developing hormonal and metabolic disorders [2].Obesity is stated to be the main elements responsible for a decrease in general testosterone levels [3].As urbanization continues to rise across Asia, it is expected that the forthcoming obesity epidemic will be predominantly concentrated in this re-gion [4,5].Extensive research has confirmed a direct link between testosterone and obesity [6].Testosterone deficit adversely affects many organs, and obesity causes a decrease in testosterone levels [7,8].Still, the duration for a decrease in androgens and clinical manifestation of hypogonadism in men can vary [9].Physiological aging in men is determined by population-specific characteristics and individual genetic traits, including genetic mutations.Such mutations can lead to genetic polymorphism, which is predominantly present in the form of single nucleotide polymorphism (SNP).Genes are believed to play a major role in reducing androgen levels.The study considered the impact of hypogonadism on various aspects of men's physiology, including functional, biochemical, and genetic levels [10,11].The metabolism of sex hormones, primarily testosterone [12], is a key aspect in most studies.The regulation of testosterone production, binding to transport proteins, catabolism, and interaction with tissues is determined by the function of several key proteins.For men's physiology, not only the synthesis of androgens is important, but also its transportation to target cells.Also, sex hormone binding protein (SHBG) can play a big role since it regulates blood correlation of total, bio-available, and free testosterone [13].Three SNP, rs12150660, rs727428, and rs10822184, are associated with testosterone levels in populations of European ancestry [3].However, genetic polymorphisms may vary across ethnic groups [14][15][16][17][18]. Particularly, the significance of the rs727428 polymorphic locus is determined by changes in the function of the marker promoters of histone proteins and increased synthesis of the ribonucleic (RNA) protein SHBG [15].Similar relationships have been established for frequent mutations in rs5934505 and rs10822184 [19] in a number of populations.However, there is no data on SHBG polymorphism in men from Central Asia and Kazakhstan.
This study aimed to determine the relationship between mutations associated with impaired synthesis of the SHBG protein (rs727428, rs5934505, rs10822184) and hypogonadism in the Kazakh population of East Kazakhstan.

MATERIAL AND METHODS
The study involved 300 men of Kazakh nationality living in the city of Semey, East Kazakhstan, Republic of Kazakhstan.The research adopted a case-control design, and the sample size was calculated using the EpiInfo calculator.Participants were men presenting clinical manifestations of hypogonadism who sought urologist advice.Individuals with a history of androgen replacement therapy.

Inclusion criteria
After laboratory confirmation of hypogonadism, all participants provided written informed consent.Participants were divided into three groups based on their conditions: Body mass index (BMI) was calculated using the following formula: BMI=weight (kilograms)/height² (meters).An online calculator for the Asian population was used: https://irm.kz/andrology-2/kalkulyator-indeksa-massy-tela/.Weighing was conducted with participants wearing only underwear and socks.Waist circumference was measured directly on the skin at the level of the navel in a standing position.Reference BMI values were as follows: • normal body weight: 18.5-24.9;• overweight: 25.0-29.9;• 1 st -degree obesity: 30.0-34.9;• 2 nd -degree obesity: 35.0-39.9;• 3 rd -degree obesity: 40.0 or higher.
General testosterone, sex hormone binding globulin (SHBG), and luteinizing hormone (LH) were tested on Architect i2000SR equipment (Abbott Laboratories, IL, USA) using commercial diagnostic kits (Abbott Laboratories, USA) according to the manufacturer's instructions.
Bioactive и Free testosterone was measured by an online calculator http://www.issam.ch/freetesto.htmdeveloped by the Hormonology Department, University Hospital of Ghent, Belgium, with inputting data for total testosterone, SHBG, and albumin.
For genetic research, we used the peripheral blood of the test subjects, collected in vacuum tubes with K2/K3 Ethylenediaminetetraacetic acid (EDTA).Isolation of genomic deoxyribonucleic acid (DNA) from blood samples was accurately performed using a commercial GeneJET Mini kit (Thermo Scientific, Vilnius, Lietuva), according to the manufacturer's instructions.The DNA concentration was assessed by Qubit 4 fluorometer (Thermo Scientific, Walthem, MA, USA).The isolated DNA was frozen and stored at -20°C.
Genotyping of 300 DNA samples, after preliminary quality and quantity checks, was carried out by real-time polymerase chain reaction (PCR) on a CFX 96 amplifier (BioRad, CA, USA) using readymade mixtures of primers and TaqMan probes, a master mix of rs754493647 gene polymorphisms, rs727428, rs5934505, and rs10822184.The total volume of 96 well plates was 25 μl, with the composition being as follows: 2x TaqMan master mix (12.50 μl), 20x master mix (1.25 μl), and DNA (11.25 μl) (20 ng) (all (p=0.09;p=0.1).This level of difference distinguished Group 1 from Groups 2 and 3, but no difference was found between the latter groups.For the rs5934505 mutation, the corresponding values were χ2=85.179for allele frequency and χ2=69.801for genotype frequency (p<0.001).Less pronounced differences were identified for the rs10822184 mutation.These differences were significant in terms of the ratio of alleles (χ2=10.092,p=0.04) but not in terms of genotypes (χ2=9.184,p=0.05).
Table 3 presents the data on the distribution of participants according to the degree of excess body weight.
Purposeful patient selection determined significant differences in the structure and average BMI between Groups 1 and 3, as well as 2 and 3.There were no differences between Groups 1 and 2 in terms of this feature, which, to a certain extent, indicates the low significance of the role of metabolic differences in the manifestation of hypogonadism between these groups.
The analysis of BMI among the study participants was conducted by assessing the distribution of patients included in the study.There was a significant difference between Groups 1-2 and 3 in terms of BMI (χ2=296.117,p<0.001).However, no significant difference in BMI was identified between Groups 1 and 2. Table 4 presents data from the hormonal status analysis.
The total, bioactive, and free testosterone content was significantly higher in both comparison groups compared to patients with hypogonadism (p<0.001;p=0.017; p=0.033).A similar pattern was identified for SHBG (p=0.012) but not for LH.The main focus was analyzing the relationship between the presence of mutations in the SHBG protein gene and the studied parameters (BMI, hormone content, and the protein itself).reagents from Life Technologies, Foster City, CA, USA).Amplification was performed for 10 min at 95°C, 50 denaturation cycles lasting 15 sec at 92°C, and annealing for 90 seconds at 60°C for all polymorphisms.

Statistical analysis
The database was created using Microsoft Excel, and statistical analyses were conducted using SPSS version 20.0 software, licensed through Semey State Medical University, Kazakhstan.The Pearson χ2 test was employed to analyze contingency tables, while differences in continuous data sets were assessed using the Kruskal-Wallis method, following established guidelines [20].A significance threshold of p<0.05 was applied to determine statistical significance and reject the null hypothesis.

RESULTS
The age distribution across all groups showed no significant differences, and the average age of the groups was not significantly different (p>0.1).Among concomitant chronic somatic diseases, arterial hypertension (χ2=13.569,p=0.002) was more prevalent in Group 1 compared to Groups 2 and 3 (Table 1).The genetic test outcomes are presented in Table 2.
The allelic structure of the studied gene did not exhibit significant deviations from the Hardy-Weinberg distribution for any of the three mutations.When analyzing the significance of differences in allele and genotype frequencies, no significant differences were observed for the rs727428 mutation in either case statistically significant (p=0.033),but there was no significant dependence for bioactive testosterone.Also, no apparent features of SHBG and LH were determined to be associated with the number of identified mutations.In Group 2, no significant relationship was observed between body weight indicators and the presence and number of SHBG protein gene mutations.The content of total testosterone in the presence of mutations was reduced and had significant differences depending on their number.The significance index in the data series was 0.023.A similar pattern was observed for the content of bioactive testosterone, with a significance level of p=0.012.At the same time, only moderate downward trends in The number of mutant alleles at all three loci was categorized into different groups: absence, one, two, three, or three or more.Gradation "three" was determined only in Group 3, where a greater number of mutant alleles were detected.
There was no significant dependence of BMI level on the presence and number of mutant alleles in the SHBG protein gene.However, there was a tendency for the BMI indicator to increase in individuals with a higher number of mutations.
The content of total testosterone in all groups tended to decrease both when the presence was detected and with an increase in the number of mutations.In Group 1, the dependence of total testosterone content on the number of mutations was The data from a number of studies has revealed that these mutations are quite common and have a unidirectional effect on the content of total testosterone.It decreases to varying degrees due to a reduction in the binding protein concentration [32].At the same time, the amount of free testosterone, which determines the intensity of its synthesis, practically does not differ in individuals with and without these mutations [19,33].This phenomenon is specified by the action of hormone synthesis regulatory systems independent of the SHBG protein and adjusted by the level of free testosterone [34].
There is also evidence of a relationship between violations of testosterone levels in the blood and the development of obesity or the presence of obesity [2,35].Low values of the indicator in young and middle-aged men in the population have been associated with a high level of BMI [23,36].However, the diagnosis of hypogonadism and decreased sexual function are not always associated with obesity.There is a category of men who maintain high fertility in the presence of a moderate increase in BMI and obesity [37].
The mentioned characteristics of genetic regulation regarding testosterone binding offer a potential explanation for this phenomenon [38].Our study found a clear relationship between the development of hypogonadism in overweight men and the content of bioactivated testosterone in the blood.Moreover, in cases of hypogonadism, this indicator was significantly lower compared to the group without elevated BMI and sexual function impairments.Conversely, these levels were even slightly higher in the group with elevated BMI but without hypogonadism.
SHBG polymorphism is a significant factor influencing testosterone concentration and can serve as an indicator for identifying men with low testosterone levels.Our data contributes to comprehending the etiology behind the early development of hypogonadism in men.
This study has some limitations, including its small sample size, which may limit the generalizability of findings.In future research, we aim to conduct a more comprehensive investigation with a larger sample to provide a more comprehensive understanding of the topic.

CONCLUSION
The frequency of mutant alleles of genes determining the intensity of SHBG synthesis in the studied Kazakh population was high and comparable to the indicators found in the studies on European populations and Southeast Asian countries.The association between mutation rates and obesity is ambigu-the presence of mutations were distinctive for the SHBG level, as well as for an increase in the LH level.
In Group 3, there was no significant relationship observed between an increase in BMI and the number of mutations in the studied gene.However, similar to the previously described groups, there was a decrease in the content of total testosterone (p=0.028) and bioactive testosterone (p=0.021)depending on the number of mutations.Differences in the series of SHBG values tended to drop, and this trend became more pronounced with an increased number of mutations in the corresponding gene (p=0.033).Conversely, there were no significant differences in LH levels, similar to the other groups of patients.

DISCUSSION
The SHBG protein is regarded as a hormone sequester crucial in controlling hormone bioavailability.A change in the concentration of SHBG can significantly affect the androgenic status of a man.In elderly and aged men, an increase in the level of SHBG in the blood is observed, which decreases as the body weight increases.
The mechanism by which the drop in SHBG levels is observed in obese patients remains debatable, but it is supposed to be the suppression of SHBG synthesis in the liver by elevated insulin concentrations [21][22][23].
The role of the testosterone-binding protein in the human body might be dual.On the one hand, the binding of this hormone determines a decrease in its biological activity [24,25], while on the other hand, it specifies the preservation of its significant concentration in the deposited form [26].
In this case, the pool of bioactive testosterone is determined by the concentration associated with blood albumin and its free fraction [27].SHBG gene polymorphism determines its differences, including discrepancies in levels or activities of key transcription factors.
Disturbances in the structure and function of the protein, as well as changes in its amount, can result in disturbances in the binding of sex hormones [28].At the same time, there is evidence of testosterone functioning in a bound species through interaction with cellular receptors [29].Accordingly, changes in the synthesis and structure of this protein can lead to multidirectional features of the total content and ratio of testosterone fractions [30].
A common feature is decreased SHBG protein content in the presence of mutations in the binding loci associated with the corresponding RNA synthesis [31].ous.Hypogonadism was found to be caused by the decrease in SHBG level and the associated total testosterone.Testosterone associated with SHBG is not bioavailable for all target tissues, and its level depends on the concentration of SHBG, which is inversely correlated with BMI and abdominal fat.The level of the bioavailable fraction of testosterone did not depend on the concentration of SHBG polymorphic forms.This phenomenon is explained by the action of hormone synthesis regulatory systems independent of the SHBG protein and adjustable by the level of free testosterone.